The main goal of our Project (3) is to develop novel nucleoside and nucleotide inhibitors directed against Alphaviruses. There is a very important need for the development of treatments against these important human pathogens. We propose to identify potent inhibitors of CHIKV and VEEV replication by performing a high-throughput screen with chemical libraries from Gilead (50,000 heterocycles and nucleoside analogs), HitCreate library from Tranzyme (50,000 macrocycles) and the Diversity Library (27,000 heterocycles). Compounds displaying activity against the two viruses will be further validated and then characterized for specificity within the Alphavirus genus, efficacy and toxicity in multiple relevant cell-types, mode of action, and generation of viral mutants. Compounds that pass this second set of filters will undergo Hit to lead optimization and SAR with subsequent retesting in secondary screens. Last of all, we will perform in vivo efficacy testing in relevant mouse and non-human primate models of alphavirus infection and disease for compounds with good bioavailability and activity. Through these studies we will develop robust antiviral agents against multiple members of the Alphavirus genus.